The present invention is related to the analysis of liquid samples with a spectrometer or photometer or other optical measuring devices. Such analyses are made typically, but not exclusively, in the molecular-biological, biochemical, inorganic-chemical, and organic-chemical and foodstuff chemical laboratory. Samples are optically analysed for instance in research, in diagnostics and in quality control. They are analysed for instance by way of absorption-, reflection-, emission-, fluorescence-, Raman- or luminescence spectroscopy in the UV-VIS or IR wavelength range. Examples for analytes to be measured are biomolecules like nucleic acids, proteins, lipids as well as inorganic or organic materials and compounds. These analytes can be measured directly or after a chemical reaction that serves for facilitating the spectrometric or photometric analysis.
The present invention is related in particular to all the applications that were mentioned by way of example above. An essential field of its application is the measurement of valuable samples in small amounts in molecular biology. Often only small amounts of sample are at hand (for instance, from less than 1 up to 5 micro-liters), because nor more material can be obtained. When diluting the samples, the measurement results would become too inaccurate due to decreased absorption. A typical application is the photometric or fluorometric measurement of nucleic acid concentrations before a PCR or real-time PCR, in order to be able to use that starting amount of the nucleic acid which is optimum for PCR. Another example is the measurement of the concentration of nucleic acids and marker substances incorporated into the nucleic acid, as well as of the marking density of marked nucleic acids derived from this, in order to be able to use the optimum amount of marked nucleic acid before beginning a micro array experiment, and to be sure that the marking density of the nucleic acid is in the optimum range.
For spectrometric or photometric analysis, liquid samples are filled into cuvettes. Standard cuvettes are suited for the insertion into the cuvette shafts of most of the current spectrometers and photometers. These cuvette shafts are also called “standard cuvette shafts” in the following. Standard cuvette shafts of usual commercial optical measuring instruments having a cross section of 12.5 mm×12.5 mm have become wide-spread. The heights of the light beam above the bottom of the cuvette shaft vary from 8.5 mm to 20 mm, depending on the type of the device. Standard cuvettes have a box-like outline, wherein the cross section and the height are matched to the dimensions of the standard cuvette shafts.
Re-usable standard cuvettes of quartz glass for small amounts of sample are marketed by the companies Hellma and Starna in particular. These ultra micro cuvettes have a layer thickness of 1 mm or more. It is very difficult to fill them without bubbles, and very sumptuous to empty and to clean them. Because the main application of the optical measurements is the measurement of very small volumes when measuring nucleic acids in the UV region, they are made of quartz glass and are particularly expensive. They must be treated with much care, because they are very expensive to buy. For the ultra micro cuvettes of quartz glass that are obtainable on the market, a minimum volume of 5 micro-liters must be used, which is too much for many applications.
Other cuvettes are marketed with the designation “Mikroliter-Messzelle” (micro-liter measuring cell). Under the product name “Tray Cell®”, the company Hellma, and under the product name “Label Guard” the company Implen markets a micro-liter measuring cell which corresponds to a standard cuvette in its dimensions, and may therefore be used in many of the today's spectrometers. The micro-liter measuring cell of the company Hellma is described in WO 2005/114146 A1. In order to make an analysis, one drop of about 1 to 2 micro-liters of the liquid to be analysed must be applied to the topside of a measuring window at a layer thickness of 0.2 mm, or at a layer thickness of 1 mm when the drop is 3 to 5 micro-liters. The measuring chamber is closed by a lid. The light beam of the measuring optics is guided from the radiation source through the sample to the sensor via beam deflections and fibre-optic light guides and via a mirror in the lid.
The micro-liter measuring cell is very sumptuous in its construction and it has a high price, and therefore it cannot always be used in an economically reasonable fashion. Moreover, it has a high apparatus-dependent intrinsic absorption of 1.3 E at 230 to 650 nm, about which the measurement range of the measuring instrument is reduced. Further, it is not possible to visually check the measuring solution in the measurement chamber after filling in the sample and putting up the lid, in order to detect disturbing bubbles, particles and erroneous pipettings that might lead to erroneous measurements. In addition it is disadvantageous that the user must clean the measuring window after use in a time-consuming way.
Under the product name “Nano Quant Plate”, the company Tecan offers a kind of collapsible micro-plate for a micro-plate reader.
Under the product name “NanoDrop®”, the company NanoDrop Technologies markets a photometer, which permits to analyse samples that have a volume of one micro-liter only. This spectrometer is described in WO 2006/086459 A2. The system envisions the direct optical measurement in a drop of liquid which is located between two horizontally aligned, planar surfaces. Alight source illuminates the sample of liquid from the side through the gap between the two surfaces. A fibre light guide runs out into the lower surface, which leads the light further to a fibre optics spectrophotometer after it has passed through the liquid sample. Thus, the sample liquid is in direct contact with the glass fibre.
In the spectrophotometer, it is disadvantageous that the optical surface can be negatively affected by certain samples. According to the operation manual of the spectrophotometer of the type NanoDrop-1000, such samples are for instance protein containing solutions. In this case, the user must manually condition anew the optical surface after repeated usage by intense, time-consuming strong rubbing. Also, strongly acidic or alkaline solutions cannot be used.
Further, the sample is in direct, open contact with the solution. Thus, dangerous substances cannot be examined with this system. However, dangerous materials, like possibly infective substances, are often used in the molecular-biological, cell-biological, biochemical and chemical laboratory. The system is not suited for these samples. Due to the open contact of the sample with the surroundings, samples may become contaminated. This may disturb the measurement. Moreover, it is not possible to re-obtain valuable samples after the measurement without the risk of contamination.
The spectrophotometer is a very expensive measuring system. It comprises a measuring unit and a PC and consumes much space. The sample may quickly evaporate and easily become contaminated, because the surface area of the open drop of liquid has direct contact to the surroundings.